Research on gene transfer of hepatocyte growth factor in hepato-spleno-surgery

肝细胞生长因子基因转移在肝脾外科中的研究

基本信息

批准号：
11671273
负责人：
YAMAGUCHI Masahiko
金额：
$ 2.43万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671273/
关键词：
adenovirus vector hepatocyte growth factor liver regeneration Insulin 肝傷害脾自家移植

项目摘要

We manufactured adenovirus vector containing hepatocyte growth factor (HGF), of which cDNA was generously provided by Dr.T.Nakamura (Biomedical Research Center, Osaka University School of Medicine), according to the protocol Dr.I Saito (Institute of Medical Science, University of Tokyo) invented.After adenovirus vector containing HGF (AxHGF) was transfected into Hela cells. we collected cultured media which contained 1000-3000 ng/ml of HGF.Bioactivity of HGF produced by AxHGF-transfected Hela cells were proved by proliferative effects on cultured vascular endothelial cells and protective effects on radical-induced hepatocyte injury.AxHGF was propagated in 293 cellsand concentrated with purification by sequential ultracentrifugation in cesium chloride step gradients. In vivo injection of concentrated AxHGF improved histological changes and serum levels of AST, ALT and LDH in the dimethylnitrosoamine-induced cirrhotic rats. Hepatic ischemia-reperfusion injury was also reduced in the AxHGF-transfected rats.Increased angiogenesis could not be observed in the splenic autotransplantation with AxHGF injection. Methods of the experiment and evaluation needed to be reconsidered.Another study utilyzing insulin gene adenovirus vector (AxIns) which we made, demonstrated that AxIns injection promoted liver regeneration and increased body weight after hepatectomy in diabetic rats. One shot injection of AxIns prolonged mean survival periods for 30 days in total pancreatectomized dogs without daily insulin, and FK506, an immunosuppressant, increased the amounts and periods of insulin gene expression by AxIns and prolonged survival periods in AxIns-transfected total pancreatectomized dogs.

根据《医学科学研究所》，我们生产了含有肝细胞生长因子（HGF）的腺病毒载体（HGF），由T. Nakamura博士（大阪大学医学院生物医学研究中心）慷慨地提供了cDNA。东京大学发明了腺病毒载体后，将HGF（AXHGF）转染到HeLa细胞中。我们收集了含有1000-3000 ng/ml的HGF的培养培养基。通过对培养的血管内皮细胞的增殖作用，证明了由AXHGF转染的HELA细胞产生的HGF的生物活性，并对自由基诱导的肝细胞损伤进行了保护。细胞和通过顺序超离心在氯化剖腹梯度梯度中浓缩。浓缩AXHGF的体内注射改善了二甲基硝基胺诱导的肝硬化大鼠AST，ALT和LDH的组织学变化和血清水平。在AXHGF转染的大鼠中也减少了肝缺血 - 再灌注损伤。在脾脏自身移植中无法通过AXHGF注射观察到血管生成。需要重新考虑实验和评估的方法。其他研究胰岛素基因腺病毒载体（Axins），我们做出的，证明轴向注射促进肝脏再生和肝切除术后糖尿病大鼠肝切除术后的体重增加。在没有每日胰岛素的总胰腺切除犬中，轴突的一射击延长了平均生存期30天，而免疫抑制剂FK506增加了轴的胰岛素基因表达的量和周期，并在轴突传递的总pancreateateateateateatpeateateatopmized狗中延长了胰岛素基因的表达和延长的生存期。